Pathogenetic Mechanisms of Putamen Dopamine Depletion in PD: We confirmed that putamen tissue from PD patients contains a buildup of DOPAL relative to DA and obtained evidence that putamen DOPAL buildup in PD is the result of two abnormalitiesdecreased vesicular uptake of cytoplasmic DA and decreased DOPAL metabolism by ALDH (Goldstein et al., J Neurochem 2013;126:591-603). To assess whether decreased vesicular uptake and decreased DOPAL detoxification build up endogenous DOPAL in catecholaminergic cells, we measured effects of vesicular uptake blockade and inhibition of ALDH and aldehyde/aldose reductase on cell and medium catechol contents in PC12 cells (Goldstein et al., J Neurochem 2012;123:932-943). These manipulations exerted additive effects on endogenous DOPAL production. In the same study we obtained evidence that intracellular DOPAL contributes to apoptosis and oligomerizes alpha-synuclein. The findings fit with decreased vesicular sequestration of cytosolic catecholamines and impaired catecholaldehyde detoxification contributing to the catecholaminergic denervation that characterizes PD. The catecholaldehyde hypothesis predicts that mice with inherited low activity of the type 2 vesicular monoamine transporter (VMAT2-Lo) should have striatal DA depletion and aging-related Parkinsonism. In an ongoing collaborative study with G. Miller (Emory) we have confirmed decreased striatal DA contents for given rates of catecholamine synthesis as indicated by tissue DOPA in VMAT2-Lo mice. The catecholaldehyde hypothesis also predicts that mice with inherited low ALDH activity should have aging-related nigrostriatal neurogeneration manifesting as Parkinsonism. In a collaborative study with R. Strong (Univ. of Texas, San Antonio) we tested this prediction in mice with knockout of ALDH1A1 and ALDH2, the two isoforms known to be expressed in substantia nigra DA neurons. Double knockout mice had congenitally increased striatal DOPAL and age-dependent deficits in motor performance, loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra, and reduced striatal DA (Wey et al., PLoS ONE 2012;7:e31522). PD patients were found to have low putamen DA:DOPA, DA:(DOPAC+DOPET+DOPAL), and DOPAC:DOPAL, compared to controls (Goldstein et al., J Neurochem 2013 Jun 21. doi: 10.1111/jnc.12345). These findings provide novel neurochemical evidence for decreased vesicular uptake of DA, a shift from vesicular uptake to oxidative deamination of cytoplasmic DA, and decreased ALDH activity in the striatum in PD, as predicted by the catecholaldehyde hypothesis. Pathogenetic Mechanisms of Myocardial Norepinephrine Depletion in PD: Since as noted above we found evidence that a shift from vesicular sequestration to oxidative deamination of cytosolic catecholamines contributes to putamen DA depletion in PD, we hypothesized that an analogous shift takes place in sympathetic nerves in the heart. If so, then in residual sympathetic nerves concentrations of the deaminated catecholamine metabolites, 3,4-dihydroxyphenylglycol (DHPG) and 3,4-dihydroxyphenylacetic acid (DOPAC), would be elevated relative to NE. We estimated rates for steps in the synthesis and disposition of catecholamines in cardiac sympathetic nerves in PD and validated the neurochemical approach in transgenic mice with low activity of the type 2 vesicular monoamine transporter (VMAT2-Lo). Myocardial NE was decreased by 97.5% in PD, associated with high tissue DHPG:NE and DOPAC:NE ratios. In VMAT2-Lo mice, NE was decreased by 96.6%, with increased deaminated metabolite:NE ratios as in PD. Decreased NE in PD was from an estimated 5-fold increase in NE turnover, a 53% decrease in NE formation, and a 74% loss of noradrenergic nerves (unpublished observations). Myocardial NE content therefore is decreased drastically in PD. The depletion reflects denervation combined with a shift from vesicular sequestration to oxidative deamination of cytosolic catecholaminesthe same abnormal pattern as in the putamen. The findings lead to the proposition that decreased vesicular uptake is part of a final common pathway in the death of catecholamine neurons in PD. Pathogenetic Mechanisms of Loss of Locus Ceruleus Noradrenergic Neurons in a PD Mouse Model: In a collaborative study with G. Miller (Emory) on neuropathologic consequences of very low VMAT2 activity, we obtained evidence for acceleration of aging-related loss of noradrenergic neurons in the locus ceruleus, the main source of norepinephrine in the brain, in VMAT2-Lo mice (Taylor et al., J Neurochem (in press)). Links between Alpha-Synucleinopathy and Catecholaminergic Denervation: Lewy bodies, intra-neuronal inclusion bodies that are prominent in brainstem monoaminergic neurons, are a pathologic hallmark of PD. Lewy bodies and associated Lewy neurites contain abundant precipitated alpha-synuclein, and alpha-synucleinopathy is etiologic in some forms of familial PD. Understanding links between alpha-synucleinopathy and catecholaminergic denervation therefore is important for elucidating the pathogenesis of PD; however, why catecholamine neurons are relatively selectively lost in synucleinopathies and why alpha-synuclein tends to precipitate in catecholaminergic neurons have been mysterious. The catecholaldehyde hypothesis straightforwardly answers these questions, by posing that alpha-synucleinopathy interferes with vesicular function and thereby increases generation of DOPAL and that DOPAL oligomerizes alpha-synuclein, converting alpha-synuclein into a toxic agent. These interactions set the stage for pathologic positive feedback loops that eventually destroy the neurons. In confirmation that DOPAL formed intra-cellularly oligomerizes alpha-synuclein, we found that exposure of alpha-synuclein-overexpressing PC12 cells to levodopa generated DOPAL and evoked alpha-synuclein dimerization and that blockade of monoamine oxidase prevented the dimerization (Goldstein et al., 2012;123:932-943). Conversely, mice over-expressing the A53T form of the alpha-synuclein gene in substantia nigra dopaminergic neurons have decreased striatal DA (J Neurosci 2012;32:9248-9264). These mice also have neurochemical evidence for a shift from vesicular uptake to oxidative deamination in striatum (unpublished observations). The Braak staging concept of Lewy body disease pathogenesis is based on post-mortem evidence for a spatiotemporal sequence of alpha-synuclein deposition, with autonomic nervous system involvement before synucleinopathy in substantia nigra neurons. A patient with primary chronic autonomic failure underwent biennial brain 18F-DOPA and myocardial 18F-dopamine scanning over four years. Low myocardial radioactivity indicated cardiac noradrenergic denervation that persisted. Striatal 18F-DOPA-derived radioactivity initially was normal, two years later was decreased subtly, and by four years was clearly decreased and accompanied by dementia and Parkinsonism. In this case, neuroimaging evidence of cardiac noradrenergic denervation and subsequent progressive striatal dopaminergic denervation provided in vivo support for Braak staging (Goldstein et al., Clin Auton Res 2012;22:57-61).